Slides Esterlizaçao

BIOTECHNOLOGY FOR ENGINEERS Core Course for B.Eng.
(Chemical Engineering) Semester I (2010/2011)

Mohamad Hekarl Uzir, MSc.,DIC.,PhD.(London)-chhekarl@eng.usm.my
School of Chemical Engineering Engineering Campus, Universiti Sains Malaysia Seri Ampangan, 14300 Nibong Tebal Seberang Perai Selatan, Penang
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Course Contents
TOPIC 1: Sterilisation

Introduction to sterilization Medium sterilization Thermal death kinetics of microorganisms Effect of sterilization on quantity of nutrients Sterilization of culture media Batch sterilization Design aspects Del factor during heating and cooling Calculation methods
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Course Contents
TOPIC 1: Sterilisation

Continuous sterilization Sterilization of air Methods of air sterilization Theory of brous lter Sterilization of fermenter
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Course Contents
TOPIC 2: Bacterial Growth

Growth curve and the phases involved Batch culture Material balance for batch culture Kinetics of batch cultureMonod Model Continuous culture Material balance for continuous culture Kinetics of continuous cultureMonod Model
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Course Contents
TOPIC 2: Bacterial Growth
Cell growth Cell growth measurement Cell countstoichiometric of cellular growth Continuous growthIdeal Chemostat
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Course Contents
TOPIC 3: Effect of Environmental Conditions
Effect of different conditions towards growth kinetics Effect on temperature Effect on pH Effect on oxygen concentration Heat generation by microbial growth
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Course Contents
TOPIC 4: Enzyme-Catalysed Reactions (Prof. Azlina)
Enzyme Kinetics Reactions involving enzymes Michaelis-Menten Model Enzyme inhibition Immobilised-Enzyme system
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Overview
Biochemical Engineering Overview
BIOCHEMICAL ENGINEERING
Upstream Processing
Downstream Processing
New Drugs & Clinical Trials
Biocatalysis
Biotransformation\ Bioconversion Fine Chemical Synthesis (precursors/ synthons) Enzyme-catalysed Transformation Whole-Cellcatalysed Transformation Two-Phase Transformation (Ionic Liquid)
Bioreactor Design Conventional Bioreactor Novel Bioreactor for Biotransformation Bioreactor with in situ Product Removal
Solid-Liquid Separation Liquid-Liquid Extraction Cell Disruption Solvent Recovery Crystallisation
Gene Modification Gene Expression Recombinant DNA Fermentation Directed Evolution
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Sterilisation
Introduction
It differentiates a biochemical process from a chemical process. Pure bacterial (or any organisms) culture requires a contamination-free environment to grow Therefore, a container use to grow this organism should be free from contaminants (eg. bacteria/fungi from other species)
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Sterilisation
Fermentation can only proceed with the following: 1. a microorganism 2. a medium 3. a fermenter 4. nutrients (other additives) 5. air, for aerobic process
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Sterilisation

A sterile environment requires all of the above Else, contaminants will dominate the whole scene which will lead to NO PRODUCT. Contamination by a foreign organism may result: 1. contamination of the nal product 2. medium would be consumed unnecessarily to support growth of contaminating organism 3. contaminated product will overweigh the desire product
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Sterilisation
Contamination by a foreign organism may result: 4. contaminated product may interfere with the recovery of the desired product 5. unsterile air in aerobic fermentation may result in the spoilage of the fermentation product.
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Sterilisation
To overcome the above problems is to carry out sterilisation using any of these methods: 1. sterilisation of medium 2. employing as pure inoculum as possible 3. sterilisation of fermenter 4. sterilisation of pipes, valves, etc. which come in contact with the fermentation process 5. sterilisation of all materials to be added to fermenter 6. sterilisation of air
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Sterilisation
To overcome the above problems is to carry out sterilisation using any of these methods (contd): 7. disinfecting the fermenter and contact parts with non-toxic disinfectant 8. maintaning aseptic conditions in the fermenter during fermentation 9. maintaning the optimum/desired pH which discourages the growth of certain contaminants/undesired organisms.
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Sterilisation
Sterilisation can be carried out using: 1. HEAT from the steam 2. RADIATION from UV light 3. CHEMICAL 4. FILTRATION
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Sterilisation
Medium Sterilisation
Medium or media (singular) is an aqueous component which consists of source of nutrients and vitamins for bacterial growth. It should be free from any organisms before it can be used for bacterial/fungi inoculation. Medium contamination can lead to: 1. other type of organism to use the nutrients 2. this can lead to changes in chemical structure of the nutrients 3. change in pH
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Sterilisation
Medium contamination can lead to (contd): 4. foam formation 5. formation of metabolic productsleads to growth of fermentation 6. alter the oxidation/reduction potential of the medium 7. destroy/alter/degrade fermentation product
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Sterilisation
Steam/heat sterilisation is the most common method used Several techniques can be applied: 1. boiling in water 2. passing live steam 3. autoclaving (in pressurised vessel) It can either be batch or continuous or HTST
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Sterilisation
Synthetic mediado not require much sterilisation compared to crude media synthetic(or dened) is a type of medium which consists of known chemicals component (phosphate, chloride, nitrate etc.) crude(or complex such as Luria-Bertani (LB)) is a type of nutrient which mainly consists of unknown amount of nutrients. The medium normally contains yeast extract, peptone and glucose/glycerol. Synthetic media may require only a small amount of heating for sterilisation
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Sterilisation
Crude media are likely to contain fungal spores and sometimes traces of bacterial cells, therefore, requires prolong heating However, excessive heating may cause protein denaturing or degrade the sugar pH should also be adjusted to neutrality (7.0) before sterilisation then adjust back to the required pH with pre-sterilised acid/alkali If special nutrient/vitamin is required during the fermentation, it can be lter-sterilised prior to inoculation
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Sterilisation
Thermal Death Kinetics of Microorganisms
Thermal heatate of destruction of microbes by r steam/moist heat it is described as;

dN = kN dt
(1)
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Sterilisation
or upon integration gives,

N0 = ln Nt
t
kdt
0
(2)
where; N = no. of live organisms present t = sterilisation time period k = rst order thermal death kinetics rate constant
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Sterilisation
The negative sign indicates as t increases, the number of organisms present, N tends to decrease Further integration of the equation leads to;
N0 = kt ln Nt
The ratio N0 is the inactivation factor while the Nt reverse of the ratio describes the survival factor. The graphical representations of the function are discussed separately The k value depends entirely on the type of cells used as well as the physiological form of the cells.
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Sterilisation
Effect of sterilisation on quality of nutrients: The interactions (between nutrients) can cause deleterious effect to the medium Discoloration (due to Milliard browning) is a common case when reducing sugar reacts with amino acids Therefore, carbohydrate components should be separated from the rest of the medium
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Sterilisation
Sterilisation of culture media: Different cells/spores have different thermal death effects which can only be described using the rst order equation as well as the temperature related equationArrhenius equation given by;
k = Ae
E RT
TEST YOURSELF: How do you relate the above equation with the given graph?
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Sterilisation
Higher activation energy, EA
ln k
A
EB EA
Lower activation energy, EB
1/T
Heat supplied
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Sterilisation
Sterilisation of culture media (contd): Sterilisation of mixed cultures with different sterilisation timeleads to different extents of viable cells Consider a mixed culture, C which consists of cultures A and B Using
N0 ln = kt Nt ln Nt ln N0 = kt
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Sterilisation
Sterilisation of culture media (contd): Plots of each culture depending on the cellular viability are as follows: 1. Figure (a): the more sensitive organism is dying in the early stages when curve C is following line A. When more amount of A is dead, the curve follows the path of B 2. Figure (b): the more sensitive organism is LESS in number, it dies rather quickly, therefore, the total number of cells will always equal to the number of cells of the resistant organismB. Hence, curve C almost follows that of B.
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Sterilisation
ln Nt
ln Nt
C C B B B
A Time (a) High propotion of sensitive culture A
(b) High propotion of sensitive cultur
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Sterilisation
Batch Sterilisation
Sterilisation can be carried out in two different congurations (i) batch and (ii) continuous For a batch methoditems to be sterilised are loaded in a sterilizer (autoclave) and steam is injected according to the desired programme and later discharge upon completion for further utilisation It is the popular technique use in most biotech processes, however, it has ONE disadvantagedestruction of nutrients
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Sterilisation
The advantages include: LOW initial cost of investment LESS chances of contamination after sterilisationsterilisation can be carried out in same vessel/fermenter itself LESS mechanical failuresince the control is carried out manually EASY handling of high proportion of solid media
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Sterilisation
Design aspects: The determination of whether a sterilisation is complete or not can be related to Arrhenius equation;
k = Ae
E RT
OR seldom written as;

d(ln k) E = dT RT 2
which upon integration give a constant k0 which later equals to A, the Arrhenius constant.
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Sterilisation
Design aspects (contd): Using the equation, a set of experiments at various temperatures and k is evaluated. This is later combined with the thermal death equation;
N0 ln = kt Nt
leads to;
E N0 RT ln = Ate Nt
(3)
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Sterilisation
Design aspects (contd): The above equation acts as a design criterion called Del factor denoted as;
N0 = ln Nt
(4)
which later gives;

= Ate
E RT
(5)
and upon rearrangement leads to;

ln t = E R 1 T + ln A
(6)
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Sterilisation
Design aspects (contd): The plot of the equation at different is given below:
ln (tsterile)
1/T (k x 103)
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Sterilisation
Design aspects (contd): Equation (6) acts as the design criterion called the Del Factor It is dened as the measure of fractional reduction in living organisms count over the initial number present The factor is based on certain heat and time regime Consider the spores of Bacillus stearothermophilus which is the most heat resistant microorganism with activation energy, E = 283 kJ/mol Arrhenius constant, A = 1 1036.2 s1
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Sterilisation
Design aspects (contd): Using the given information and Nt = 103 , the factor can be easily determined; with
Nt = 103
and
N0 = ln Nt
if the unsterile culture contains N0 = 1011 viable cells, hence

1011 = ln 3 = ln 1014 = 32.2 10
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Sterilisation
Design aspects (contd): The value 32.2 is considered as the overall factor. Cells normally destroy at 121 C temperature and 0.1 MN/m2 gauge This is called the holding period since the medium is held at 121 C Some of the cells will also get destroyed during the heating up to 121 C which is called the heating period Some will get destroyed during the cooling period from 121 C to room temperature
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Sterilisation
Design aspects (contd): This can be simplied into;

overall = heating + holding + cooling
factor during heating and cooling: From Equation (6), it is obvious that when the temperature increases, factor also increases It is possible to get at any temperature holding period But temperature is not constant during heating-up and cooling-down periods
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Sterilisation

factor during heating and cooling (contd): Using;

N0 = ln = Nt
kdt
0
(7)
With the above equation, the heating-up and cooling-down periods can be determined, provided the temperature and time is known However, it is highly unlikely to describe the function of t an T in the form of linear, hyperbolic or exponential Therefore, graphical integration is advisable
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Sterilisation

factor during heating and cooling (contd): An experiment to get a prole of temperature w.r.t time is conducted A graph of time, t against temprature, T is then plotted The temperature, T1 during a time, t1 is noted as an average temperature at the beginning of t1 until the end of t1 i.e T1 is marked at the midpoint of t1 Consider the culture of B. stearothermophilus with Activation energy, E = 283 kJ/mol and Arrhenius constant, A = 1 1036.2 s1
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Sterilisation

factor during heating and cooling (contd): Therefore,

1 = k1 t 2 = k2 t 3 = k3 t
etc. which gives the summation of individual of;

total = 1 + 2 + 3 +
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Sterilisation

factor during heating and cooling (contd): Hence,

n
heating =
i=1
(8)
Then at the given temperature can be determined by substituting in the Arrhenius equation
k = Ae
E RT
A new graph can be plotted with Arrhenius values, k against time, t and the area under the graph can be calculated
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Sterilisation

factor during heating and cooling (contd): Refer to the separate table of time-temperature data of batch sterilisation. during heating period from 100-121 C within the time of 1-15 min is 8.7 or
heating = 8.7
Holding time (121 C): 1. Holding time can be calculated using

overall = heating + holding + cooling
when overall is determined from the given Nt = 103 and N0 = 1011
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Sterilisation

factor during heating and cooling (contd): Holding time (121 C) (contd): 2. If
cooling = 10
thus,
32.2 = 8.7 + holding + 10
hence,
holding = 13.5
3. The specic death rate k = 2.54 min1 ,

13.5 t= = = 5.3min k 2.54
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Sterilisation

factor during heating and cooling (contd): Alternative methodRichards Rapid Calculation 1. Destruction of spores is at a considerable amount only at temperature > 100 C 2. Heating period is only become effective beyond 100 C up to 121 C 3. Similarly, for cooling period, the destruction of spores will only take place until the temperature drops up to 100 C (below which, it will not be signicant) 4. Using the same conditions of B. stearothermophilus Arrhenius constant, A = 9.51 1037 min1 Activation energy. E = 283 kJ/mol
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Sterilisation

factor during heating and cooling (contd): Alternative methodRichards Rapid Calculation 5. The factor at 100 C is neglected and at other temperatures are based on the value at the previous temperature which occurred 1 min earlier (since temp. raise is 1 C/min)
(T ) = (T 1) + k(T ) 1.0
6. k(T ) is the thermal sterilisation occurred at temp. T in 1 min.
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Sterilisation

factor during heating and cooling (contd): Alternative methodRichards Rapid Calculation 7. The method bears on two assumptions: spores destruction takes place only beyond 100 C cooling & heating cycles are linear 8. When temp. during heating rises in t1 min (for 25 min) from 100 C to 121 C instead of 21 min, heating will be proportionately changed;
heating t = 9.601 21
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Sterilisation

factor during heating and cooling (contd): Alternative methodRichards Rapid Calculation Therefore,
25 (heating ) = 9.601 = 11.43 21
9. If the cooling takes place rapidly in 15 min instead of 21 min, thus,

cooling 15 = 9.601 = 6.86 21
10. Once these two values are determined, the holding factor, holding can be calculated.
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Sterilisation
Continuous Sterilisation
There will be continuous inow and outow of materialbest if capacity of operation is high Advantages include: 1. capacity/throughput can be higher 2. medium quality can be better maintained 3. can be controlled (avoid human error) 4. cost of running/operationLESS 5. short sterilisation time 6. the only option for HTST operation 7. holding capacity of steamLESS 8. ease in process scale-up
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Sterilisation
Disadvantages include: 1. HIGH initial capital investment 2. aseptic transportation along the line Two method of continuous sterilisation: 1. continuous plate heat exchange 2. continuous steam injection and ash cooling
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Sterilisation
Continuous plate heat exchange
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Sterilisation
For a continuous plate heat exchange 1. Incoming unsterile medium is preheated by heat exchanger with the outgoing sterile medium. 2. Subsequently, it is heated with steam in the heat exchanger and passed through the holding section 3. The holding section maintains the medium according the required holding time requirement, ow rate of medium and length of holding section
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Sterilisation
Continuous steam injection and ash cooling
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Sterilisation
For a continuous steam injection and ash cooling 1. Steam is directly and continuously injected along with the medium 2. Therefore, heating time and heating section are negligible 3. Holding time is based on the length of the holding pipewhere sterilisation is taking place 4. Steam and sterilised medium under pressurepassed through the expansion valve into vacuum chambersteam is removed under vacuum
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Sterilisation
For a continuous steam injection and ash cooling (contd) 5. Sterile medium passes into preheat exchangergive out heat to the incoming unsterile medium which then passes through the cooling zone
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Sterilisation
Sterilisation of Air
Air is important as a source of oxygen in aerobic fermentation For large scale fermentation, air need to be efciently sterilised For a cubic metre of air contains approx. 4 103 20 103 particles with max. possibility of 12 103 . The dust particles are approx. 0.6 microns
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Sterilisation
Methods of air sterilisation: 1. Heating 2. UV rays OR other electromagnetic waves 3. germicidal spray 4. by ltration
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Sterilisation
Heating air is possible BUT not economicaldue to its POOR thermophysical properties (lower heat transfer coeff.) UV ray is an effective technique in killing air-borne microbesonly applicable in small area Germicide can also reduce the amount of bacteria via spraying with phenol, ethylene oxide or formalincan sterile air in a small size room
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Sterilisation
Filtration is an effective method and practicallter is used to remove microorganisms form the air provided that: pores of a lter need to be smaller than the size of microbesabsolute lters pore size is bigger than the size of microbesbrous lter (cotton, glass-wool, slag, steel-wool etc.)
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Sterilisation
Theory of brous lter: The mechanisms applied are: inertial impaction, interception, diffusion, settling by gravitational force and electrostatic force The comparisons are tabulated and given separately
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Bacterial Growth
Batch Culture
Batch culture requires enough nutrient to maintain the growth

Exponential Lag phase growth phase (b) Number of cells (g/l) (c) Stationary phase Death phase
(a)
Time (min)
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Bacterial Growth
The gure shows an increase of cell at the start of the cultivation (fermentation) process Due to the presence of enough nutrient for the cell to grow The amount of nutrient decreases as it being consumed by the cell. Other side products such as carbon dioxide or ethanol is also formed simultaneously.
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Bacterial Growth
In batch cultures, the cell properties such as; size of cells internal nutrient metabolic function vary considerably during the above growth phases. No apparent increase of the amount of cell at the start of cultivation, this is termed as the lag phase.
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Bacterial Growth
After this period (can be between 10 to 15 mins) the number of cells increases exponentially thus, this stage is called the exponential growth phase; the cell properties tend to be constant last for a short period of time The next stage is the stationary phase where the population of cell achieves it maximum number. This is because: all nutrient in the closed system has been used up by the cell. lack of nutrient will eventually stop the cell from multiplying.
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Bacterial Growth
The rate of cell growth is given by;

rX = x
where rX is the volumetric rate of biomass production with units such as kg.m3 .s1 , x is the viable cell concentration with units of kg.m1 and is the specic growth rate with units of s1 .
by following the above equation, the growth is said to follow the rst-order autocatalytic reaction.
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Bacterial Growth
Similarly, the rate can be written as;

rX dx = dt
and thus;
dx = x dt
which upon rearrangement gives;

1 dx = x dt
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Bacterial Growth
Integrating the equation with the initial conditions of (x, t) = (x0 , 0) gives;
x = x0 et
Taking natural log on both sides of the equation leads to;

ln x = ln x0 + t
A plot of ln x versus time, t gives straight line with a slope .
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Bacterial Growth
Since the equation is only valid if is unchanging, therefore the plot can be used to asses whether the specic growth rate is constant for a particular cell growth. Cell growth rates are normally expressed in terms of doubling time, td . When x = 2x0 at t = td thus the equation becomes;
2x0 = x0 etd etd = 2
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Bacterial Growth
Taking natural log on both sides;

td = ln 2
or
ln 2 td =
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Bacterial Growth
The relationship of cell growth and substrate/nutrient concentration can be expressed in terms of Monod Model given by;
max [S] = KS + [S]
The function gives a hyperbolic curve upon plotting the data point.
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Bacterial Growth
The nal stage of cell cultivation is the death phase. The decrease of the number of cell occurs exponentially which happens when the cell breaks open (lysed). The rate of death normally follows the rst-order kinetics given by;
dN = kd N dt
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Bacterial Growth
upon integration leads to

N = Ns e
kd t
where Ns is the concentration of cells at the end of the stationary phase and at the beginning of the death phase and kd is the rst order death rate constant.
Both stationary and death phase, it is important to recognise that there is a distribution of properties among the cells in a population. A summary of the different phases of cell growth is given in the table overleaf.
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Bacterial Growth
Growth phase Lag
Rate of growth Zero
Comments Inoculum adapting with the changing condition (temperature, pH)
Acceleration Exponential
Increasing Constant
Trivial Population growth changes the environment of the cells
Retardation Stationary
Decreasing Zero
The effect of changing conditions appear One or more nutrients are exhausted to the threshold level of the cell
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Bacterial Growth
Growth phase Decline
Rate of growth Negative
Comments The duration of stationary phase and the rate of decline are strongly dependent on the kind of organisms
Death phase
Negative
Cells lyse due to lack of nutrient
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Bacterial Growth
The balance of a batch reactor is given by the rate of accumulation of product equals to the rate of formation of the product due to chemical reaction or can be simply written as;
d (VR c) = VR r dt dc = r dt
(9)
where c is the amount of the component and r is the reaction rate. VR in the rst line of the equation is the total volume of the culture in the reactor.
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Bacterial Growth
Sterile air or oxygen
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Bacterial Growth
The balance of a batch reactor is given by the rate of accumulation of product equals to the rate of formation of the product due to chemical reaction or can be simply written as;
d (VR c) = VR r dt dc = r dt
(10)
where c is the amount of the component and r is the reaction rate. VR in the rst line of the equation is the total volume of the culture in the reactor.
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Bacterial Growth
Continuous Culture
In a continuous culture system, nutrients are supplied to the cell at a constant rate. To maintain a constant volume of biomass in the reactor, an equal volume of cell culture is removed. This will allow the cell population to reach a steady-state condition. The reactor conguration of a continuous process is given overleaf.
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Bacterial Growth
Sterile air or Nutrient Inlet oxygen
Biomass Outlet
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Bacterial Growth
The air is pumped into the culture vessel through a sterile lter. Bubbling of air provides: supplying air for the growth of aerobic culture it also circulate and agitate the culture pressurise the head space of the culture vessel such that to provide a force during the removal of the media (and cells) from the vessel for analysis (OD, cell viability etc.). it is highly difcult to control the delivery of the nutrient and the removal of the cell so that equal amounts of medium is maintain in the vessel.
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Bacterial Growth
This can be tackled by changing the conguration of the reactor into a semi-continuous or fed-batch type reactor. The rate of ow of medium into a system of continuous culture is known as the dilution rate. When the number of cells in the culture vessel remains constant over time, the dilution rate is said to equal the rate of cell division in the culture, since the cells are being removed by the outow of medium are being replaces by an equal number through cell division in the culture.
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Bacterial Growth
Similar to that of the batch cultivation, the material balance for a continuous culture can be written as;
d (VR c) = Fo co Fi ci VR r dt
(11)
in order to maintain the volume within the vessel;

Fi = Fo = F
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Bacterial Growth
Thus,
d (VR c) = F (co ci ) VR r dt dc F dVR = (co ci ) r c dt VR dt
(12)
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Bacterial Growth
For a reactor without a recycle system,

dVR =0 dt
therefore,
dc F = (co ci ) r dt VR
(13)
let the term leads to,
F VR
denote as D, the nal equation

dc = D(co ci ) r dt
(14)
where D is the dilution rate of a CSTR cultivation system.
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Bacterial Growth
Batch growth
Material balance: consider the cell concentration in the fermenter;

d (VR x) = VR rx dt dx VR = VR rx dt
since r =
max [S] , KS +[S]
thus;
max [S] KS + [S]
dx = dt
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Bacterial Growth
Batch growth
Material balance: at steady-state,
dx dt
= 0 thus,
max [S] =0 KS + [S]
this clearly shows that when there is no growth in the bioreactor, there will be no cell to divide, thus no new cells to be produced.
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Bacterial Growth
Batch growth
Material balance: consider the substrate/nutrient concentration in the fermenter;

d (VR [S]) = VR rx dt
at steady-state condition, [S] = 0 as well.
d[S] dt
= 0 which leads to
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Bacterial Growth
Continuous growth
Material balance: the balance on the continuous stirred-tank fermenter/reactor is similar to that of the batch, provided that there are inlet and outlet to and from the fermenter respectively. consider the cell concentration, x;
d (VR x) = Fi xi Fo xo + VR rx dt
but
rx = x
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Bacterial Growth
Continuous growth
Material balance: thus;

d (VR x) = Fi xi Fo xo + VR x dt
upon expansion and let =
max [S] KS +[S]
gives,
x
dx = F i xi F o xo + V R VR dt
max [S] KS + [S]
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Bacterial Growth
Continuous growth
Material balance: upon rearranging;

dx Fi Fo = xi xo + dt VR VR
max [S] KS + [S]
for a constant volume in a bioreactor,

Fi = Fo = F
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Bacterial Growth
Continuous growth
Material balance: thus,

F F dx = xi xo + dt VR VR max [S] KS + [S] x
F let VR = D, which represents the dilution rate of the fermenter, hence,
dx = Dxi Dxo + dt
max [S] KS + [S]
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Bacterial Growth
Continuous growth
Material balance: at a steady-state condition,

Dxi Dxo +
dx dt
= 0; x=0
max [S] KS + [S]
since the cells must be grown in a sterile environment, therefore;

xi = 0
and
xo = x
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Bacterial Growth
Continuous growth
Material balance: then the equation becomes;

Dx = max [S] KS + [S] x
D = max
[S] KS + [S]
where;
Dmax =
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Bacterial Growth
Continuous growth
Material balance: at a washout steady-state, as the dilution rate, D of the continuous fermenter increases, the concentration of substrate, [S] also increases, where
D > Dmax
at x = 0.
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Bacterial Growth
Continuous growth
Material balance: the feed substrate, [S] will be such that

[S] KS
and Dmax becomes approximately equals to the maximum specic growth rate, max ,
Dmax max
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Bacterial Growth
Continuous growth
Material balance: solving for the substrate concentration leads to;

KS D [S] = max D
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Bacterial Growth
Continuous growth
Material balance: consider the substrate concentration, [S];

d 1 (VR [S]) = Fi [S]i Fo [S]o VR rx dt YX/S
where YX/S is dened as;

YX/S mass of biomass/cells produced = mass of substrate used
EKC271 p. 98/16
Bacterial Growth
Continuous growth
Material balance: expanding and rearranging the terms gives;

d[S] 1 = D([S]i [S]o ) dt YX/S
max [S] KS + [S]
at steady-state condition,
D([S]i [S]o ) 1
d[S] dt
= 0, therefore, x=0
YX/S
max [S] KS + [S]
EKC271 p. 99/16
Bacterial Growth
Continuous growth
Material balance: let the outlet substrate concentration, [S]o = [S] therefore,
D([S]i [S]) 1 YX/S max [S] KS + [S] x=0
and upon substitution of the term [S] into the above equation gives;
x = YX/S KS D [S]i max D
EKC271 p. 100/16
Effects of Environment on Cell Growth
There are 3 main environmental factors that can give effect to the cell growth; 1. Temperature 2. pH 3. Oxygen Effect of Temperature Temperaturea can change the conguration of cell constituents, especially proteins and membrane components. There is a 2-fold increase in the specic growth rate, for every 10 C rise in temperature. For certain type of cells the optimal temperature is listed below:
Courtesy of Dr. W.S. Long
EKC271 p. 101/16
Effect of Temperature psychrophiles (Topt < 20 C) Bacteria that grow at temperature in the range of -5 to 30 C. Optimum temperatures between 10 to 20 C. Microbes have enzymes which catalyse best when the conditions are cold. Cell has membranes that remains uid at these lower temperatures.
EKC271 p. 102/16
Effect of Temperature psychrophiles (Topt < 20 C) Examples of this type of organism: algae that live near the poles of the Earth at temperature below 0 C, bacteria that spoil milk, meat, vegetables and fruits even when they are stored in a fridgeit only slows down the the rate of spoilage of food and cannot stop the growth of these microbes.
EKC271 p. 103/16
Effect of Temperature mesophiles (Topt = 20 50 C) Microbes that grow at optimal temperatures in the range of 20 to 40 C. These type of organisms can be found in warm-blooded creatures e.g. humans. Pathogenic bacteria is one of the kind as well as symbiotic bacteria.
EKC271 p. 104/16
Effect of Temperature thermophiles (Topt > 50 C) Bacteria that live at temperatures exceed 50 C. It can tolerate at very harsh conditions such as decomposing materials, hot springs (temp. between 80 to 85 C) and deep in the oceans by thermal vents bubbling up from the hot rocks below the Earths crust.
EKC271 p. 105/16
Temperature ( C) Group Thermophiles Mesophiles Psychrophiles Minimum Optimum Maximum
40 to 45 10 to 15 -5 to 5 -5 to 5
55 to 75 30 to 45 15 to 18 25 to 30
60 to 80 35 to 47 19 to 22 30 to 35
Obligate Facultative
EKC271 p. 106/16
Above the temperature given above, the growth rate decreases and thermal death may occur. When the cells cannot sustain high temperature, thermal death rate exceeds the growth rate i.e. viable cells will drop. According to the Arrhenius equation;
= Ae
Ea RT d RT E
kd = A e
(15)
EKC271 p. 107/16
A typical values for Ed for thermal destruction of microorganism are high, small increase of temperature have a signicant effect on kd and the rate of death.
Ea : Ed :
Activation energy for growth Activation energy for thermal death
Temperature also affects product formation and yield coefcient.
EKC271 p. 108/16
The optimal temperature for growth and product formation differ; when T > Topt , the maintenance requirement of cell increases. ms or mp increases with increasing temperature with value of E between 15 to 20 kcal mol1 and thus decreases YX/S .(ms and mp are the maintenance coefcient for substrate and product respectively).
EKC271 p. 109/16
Temperature also affects the rate limiting step of biochemical mechanisms; during fermentation, the rate of biochemical reaction increases at higher temperature (reaction rate higher than the diffusion rate). therefore, diffusion becomes the rate limiting step. This is normally occur in immobilised cell system with pore diffusional resistance. Molecular diffusion: E = 6kcal mol1 Biochemical reaction: E = 10kcal mol1 diffusional limitations must be carefully considered at high temperature.
EKC271 p. 110/16
The plot of growth rate versus temperature of the group of microorganisms given in the previous Table is shown in Figure 1.
-1 Growth rate, (time )
Moderate thermophile Mesophile Psychrophile Extreme thermophile bacterium ? Extreme thermophile archaeon
10
20
30
40
50
80 60 70 O Temperature, ( C)
90
100 110
120
Figure 1: Growth rate versus temperature for ve environmental classes of procaryotes.
EKC271 p. 111/16
Effect of pH The inuence of pH on cellular activity is determined by the sensitivity of the individual enzymes to changes in the pH. Enzymes are normally active only within a certain pH interval and the total enzyme activity of the cell is therefore a complex function of the environmental pH. Consider the inuence of pH on a single enzyme, which is taken to represent the cell activity.
EKC271 p. 112/16
Effect of pH
-H+ [E] +H+ [E]+H+ -H+ [E]2-
Figure 2: Enzyme forms with changing of pH.
EKC271 p. 113/16
Effect of pH Where [E] is taken to be the active form of enzyme while the two other forms are assumed to be completely inactive, with K1 and K2 being the dissociation constants for the free acids [E] and [E] respectively. The fraction of active enzyme [E] is calculated to be;
[E] = [E]tot 1+ 1
[H + ] K1
K2 [H + ]
(16)
EKC271 p. 114/16
Effect of pH The enzyme activity is taken to be

k = ke [E] .
If the cell is determined by the activity of the enzyme considered above, the maximum specic growth rate, max becomes;
max = k[E]tot 1+
[H + ] K1
K2 [H + ]
(17)
EKC271 p. 115/16
Effect of pH This model has been found to t well with the specic activity data for a few microorganisms and the tting for E. coli cell as well as values of the tted parameters is given in Figure 3 and Table 1 respectively.
EKC271 p. 116/16
Effect of pH
2.0 -1 Specific growth rate, (hr )
1.5
o 37 C
1.0
o 27 C
0.5 04
7 pH
10
EKC271 p. 117/16
Effect of pH Parameter
k K1 K2
27 C 37 C 0.82 1.90 1.5105 5.0106 1.0109 3.0109
Table 1: Parameter values for the tting of equation (17 to the specic growth data).
EKC271 p. 118/16
Effect of pH The range of pH over which the microorganism grows is dened by the 3 main categories; 1. minimum pH: below which the microorganisms cannot grow 2. maximum pH: above which the microorganisms cannot grow 3. optimum pH: at which the microorganisms grow best.
EKC271 p. 119/16
Effect of pH For most bacteria there is an orderly increase in growth rate between the minimum and the optimum Orderly decrease in growth rate between the optimum and the maximum pHreecting the general effect of changing [H + ] on the rates of enzymatic reaction as shown in Figure 120.
EKC271 p. 120/16
Effect of pH
Acidophile -1 Growth rate, (time ) Neutrophile Alkaliphile
More acidic
7 pH
More basic
EKC271 p. 121/16
Effect of pH Microorganisms that grow at an optimum pH well below neutrality (7.0) are called acidophiles. Those that grow best at neutral pH are called neutrophiles and those that grow best under alkaline conditions are called alkaliphiles. Obligate acidophiles such as Thiobacillus species require a low pH for growth. This is due to their dissolving membranes and the cells lyse at neutrality. Several genera of Archaea such as Sulfolobus and Thermoplasma are obligate acidophiles.
EKC271 p. 122/16
Effect of pH A few types of procaryotes are given in Table 123.

pH Organism Thiobacillus thiooxidans Sulfolobus acidocaldarius Bacillus acidocaldarius Zymomonas lindneri Lactobacillus acidophilus Staphylococcus aureus Escherichia coli Minimum 0.5 1.0 2.0 3.5 4.04.6 4.2 4.4 Optimum 2.02.8 2.03.0 4.0 5.56.0 5.86.6 7.07.5 6.07.0 Maximum 4.06.0 5.0 6.0 7.5 6.8 9.3 9.0
EKC271 p. 123/16
Effect of pH
pH Organism Clostridium sporogenes Erwinia caratovora Pseudomonas aeruginosa Thiobacillus novellus Streptococcus pneumoniae Nitrobacter sp. Minimum 5.05.8 5.6 5.6 5.7 6.5 6.6 Optimum 6.07.6 7.1 6.67.0 7.0 7.8 7.68.6 Maximum 8.59.0 9.3 8.0 9.0 8.3 10.0
EKC271 p. 124/16
Effect of Oxygen Dissolved oxygena (DO) is an important substrate in an aerobic fermentationslimiting substrate, since O2 is sparingly soluble gas in water (7p.p.m at standard temperature and pressure: 25 C and 1atm). When oxygen is rate limiting, the specic growth rate, varies with DO. Below a critical oxygen concentration, the growth approaches a rst-order rate. Above the critical oxygen concentration, growth rate becomes independent of DO.
Courtesy of Dr. W.S. Long
EKC271 p. 125/16
Effect of Oxygen When dissolved oxygen level is below the critical level, then the oxygen concentration is a growth rate limiting, thus, another medium becomes the growth extent limiting. This can be seen in Azotobacter vivelandiiat dissolved oxygen of 0.05mgl1 , the growth rate of the organism is 50% of its maximum, even if large amount of nutrient (glucose) is present.
EKC271 p. 126/16
Effect of Oxygen This, however, does not affect the amount of cells formed since the cells will keep growing whenever there is enough oxygen dissolved. The critical oxygen concentration varies with different organisms; bacteria and yeast: 510% mold cultures: 1050% The growth extent or the mass of cells formed depends on the amount of glucose, on the other hand, the growth rate depends on the amount of oxygen dissolved, DO.
EKC271 p. 127/16
Effect of Oxygen The transfer of oxygen from gas bubbles to cells is limited by oxygen transfer through liquid lm surrounding the gas bubbles;
NO2 = kL a(C CL )
where NO2 is the oxygen transfer rate (OTR) with the units of mgO2 l1 h1 and; kL : O2 transfer coefcient (cmh1 ) a: gasliquid interface area (cm2 cm3 ) kL a: volumetric O2 transfer coefcient (h1 ) C : saturated dissolved oxygen (mgl1 ) CL : actual dissolved oxygen (mgl1 )
EKC271 p. 128/16
Effect of Oxygen Oxygen Uptake Rate (OUR) Oxygen uptake rate is given by;
X OU R = qO2 X = YX/O2
(18)
where; qO2 : specic rate of O2 consumption (mgO2 g1 h1 ) dcw YX/O2 : oxygen yield coefcients (gdcw g1 ) O2 X: cell concentration (gdcw l1 )
EKC271 p. 129/16
Effect of Oxygen Oxygen Uptake Rate (OUR) When oxygen is the rate limiting step; rate of oxygen consumed is equal to the rate of its being transferred, and assuming that there is no maintenance requirement for oxygen compared to cell growth. Therefore;
OU R = NO2 X = kL a(C CL ) YX/O2
(19)
EKC271 p. 130/16
Effect of Oxygen Oxygen Uptake Rate (OUR) since the terms (X ) is the rate of cell growth with respect to time, hence;
dX = kL a(C CL )YX/O2 dt
(20)
EKC271 p. 131/16
Effect of Oxygen Oxygen Uptake Rate (OUR) The rate of cell growth varies linearly with the amount of dissolved oxygen, DO. Thus, the concentration of oxygen in any fermentation medium should be maintained in order to obtain a stable cell growth. This can be established by; using a supply of oxygen-enriched air using pure oxygen under atmospheric pressure between 2 to 3 atm.
EKC271 p. 132/16
Heat Generation by Microbial Growth Chemical reaction that occurs within the cells produces energy which is released as heat. Cellular heat production is primarily the result of energy and growth metabolism which consequently makes the heat generated from the cells to be approximately proportional to the energy in utilising substrate.
EKC271 p. 133/16
Heat Generation by Microbial Growth the yield factor due to the heat produced, Y can be written as;
1 (gcell gsubstrate ) Ys Y (gcell kcal1 ) = 1 (Hs Ys Hc ) (kcal gsubstrate )
(21)
It is derived based on the approximate energy balances over the two different pathways shown in Figure 4
EKC271 p. 134/16
Heat Generation by Microbial Growth The predominant oxidant is oxygen, the heat generation Hs per gram of substrate completely oxidised minus Ys Hc , the heat obtained by combustion of cells grown from the same amount of substrate, will reasonably approximate the heat generation per gram of substrate consumed in the fermentation which produces cells, H2 O and CO2 .
EKC271 p. 135/16
Heat Generation by Microbial Growth

(I) Total combustion O2 + CO2 + SUBSTRATE Hs kcal/g substrate oxidised H2O
(II) Respiration yielding cellular material
(III) Combustion of cellular material
H2O
CO2
MICROBIAL CELLS
Figure 4: Approximate heat balance in substrate consumption.
EKC271 p. 136/16
Heat Generation by Microbial Growth If there are no experimental data on the energy as well as the compounds used, the heat of combustion can be estimated using the energy obtained from the transfer of electrons from a compound that has reductance degree denoted by s to a compounds such as carbon dioxide or methane which has zero reductance degree. This gives a function of
Ks
where K is within the range of 26 to 31kcal/(electron equivalence).
EKC271 p. 137/16
Example 2: Estimate the heat of combustion of Pseudomonas uorescens growing in glucose medium.
EKC271 p. 138/16
Answer 2: The reaction for cell combustion is given as;

CH1.66 N0.20 O0.27 (CELLS) + 1.28O2 CO2 + 0.10N2 + 0.83H2 O
Assuming that such a reaction produces carbon dioxide, water and nitrogen. By assuming that the heat of combustion of oxygen is 104kcal per mole of O2 , the heat released by combustion of bacteria can be estimated using the inverse of equation (21);
EKC271 p. 139/16
Answer 2:
1 Y (1.28)(104) [12 + (1.66)(1) + (0.02)(14) + (0.27)(16)] (mol O2 )(kcal mol1 O2 ) g = 6.41kcal g1
EKC271 p. 140/16
Answer 2: In an actual dry cells measurement, the weight includes about 10% of ash, therefore, the heat of combustion of cell, Hc is only 90% of the value calculated above, i.e. approximately 1 5.8kcal gdcw .
EKC271 p. 141/16
Heat Generation by Microbial Growth It can be seen that group of hydrocarbons produces more heat compared to the partially oxygenated species, for instance, Y (CH4 ) < Y (CH3 OH) and Y (n alkanes) < Y (glucose). The comparison of the growth factors between various bacteria is tabulated in the next slide.
EKC271 p. 142/16

Substrate Ys , (gcell gsubstrate ) Malate Acetate Glucose equivalents, (molasses, starch, cellulose) 0.51 1.47 0.42 0.34 0.36 YO 2 , (gcell gO2
consumed )
Y , (gcell kcal) 0.30 0.21
1.02 0.70
EKC271 p. 143/16

Substrate Ys , (gcell gsubstrate ) Methanol Ethanol Isopropanol n-Parafns Methane 0.42 0.68 0.43 1.03 0.62 YO 2 , (gcell gO2
consumed )
Y , (gcell kcal) 0.12 0.18 0.074 0.61 0.061
0.44 0.61 0.23 0.50 0.20
EKC271 p. 144/16
Heat Generation by Microbial Growth The heat produced from cellular growth can also be related to the Gibbs free energy G. Some of the free energy present in the substrate dissipates to the surrounding environment. This is apparent in an aerobic processes, the heat generated may be substantial and to keep the temperature constant, bioreactors are equipped with either external or internal cooling facilities.
EKC271 p. 145/16
Heat Generation by Microbial Growth The basis for thermodynamic calculations is the denition of Gibbs free energy in the ith reaction component;
Gi = G0 + RT ln(ci ) i
(22)
Where G0 is the Gibbs free energy at standard i conditions and ci is the concentration of the reaction component in moles per litre. In dealing with microbial growth, only free energy of certain components are required/interested thus, arbitrary energy level is introduced.
EKC271 p. 146/16
Heat Generation by Microbial Growth This is done by assigning values for the standard Gibbs free energy level of CO2 , H2 O and molecular nitrogen, N2 to zero. This reference point is chosen since no living systems can have Gibbs free energy for growth from combustion of any of these 3 compounds. Equation (22) changes to;
Gci = G0 + RT ln(ci ) ci
(23)
EKC271 p. 147/16
Heat Generation by Microbial Growth Where the subscript c refers to combustion. Using the given equation for combustion, the change of Gibbs free energy for intracellular reactions, J can be calculated;
Gc,j =
N i=1
ji Gci +
L i=1
ji Gci +
M i=1
ji Gci
(24)
j = 1, . . . , J
EKC271 p. 148/16
Heat Generation by Microbial Growth Where N , L and M refers to substrate, biomass and metabolic product respectively, while , and denes the stoichiometric coefcients of a particular growth equation. It follows the conditions: if Gc,j < 0, the reaction runs spontaneously in the forward direction. if Gc,j = 0, the reaction is in equilibrium. To calculate the energy dissipation, the last term in equation (23) can be omitted since its contribution to the overall change in free energy in a reaction is negligible.
EKC271 p. 149/16
Heat Generation by Microbial Growth The only standard free energies reduce equation (24) into;
G0 =Dj = c,j
N i=1
ji G0 + ci
L i=1
ji G0 + ci
M i=1
ji G0 (25) ci
With Dj representing the amount of energy dissipated to the surrounding environment when j th reaction proceeds. This is given by;
0 0 G0 = Hc,j T Sc,j c,j
(26)
EKC271 p. 150/16
Heat Generation by Microbial Growth Where H 0 represents the enthalpy of reaction c,j which equals to the generation of heat for the reaction. Similar to equation (25), the enthalpy balance for j th reaction can be set up as;
0 Hc,j =Qj = N i=1 0 ji Hci + L i=1 0 ji Hci + M i=1 0 ji Hci ;
j=1,..
(27)
With Qj representing the amount of heat generated by the j th reaction.
EKC271 p. 151/16
Heat Generation by Microbial Growth Multiplying this equation with the rate of the individual reactions, the specic rate of the heat generation in each intracellular reaction can be found and therefore the total specic heat generated by the growing cells by adding all the specic rates of all reactions;
N J M
rQ =
j=1
Qs,j rs,j +
j=1
Qj rj +
j=1
Qp,j rp,j
(28)
EKC271 p. 152/16
Heat Generation by Microbial Growth For reactions involving transport of species across cellular membrane such as substrate diffusing in and product diffusing out of cells, they do not contribute to the overall heat generation and thus the above equation reduces into;
J
rQ =
j=1
Qj rj
(29)
Equations (29) or (28) and (27) are important especially in estimating the amount of heat generated by growth processes.
EKC271 p. 153/16
Heat Generation by Microbial Growth A correlation was then proposed by Roels in 1983 to determine the heat of combustion of several compounds and it is given by;
0 Hci = 115 i
(30)
With the units of kJ per C-mole and is dened i as the degree of reduction of the ith compounds calculated on the basis of N2 being the nitrogen source; i.e. the multiplier for nitrogen N is zero. in the above equation is calculated using; i
= 4 + i 2bi . i
EKC271 p. 154/16
Example 3: Calculate the heat generated during the growth of Bakers yeast (Saccharomyces cerevisiae) in two different conditions; 1. aerobic growth with stoichiometric equation (without the formation of ethanol) in a dened medium;
0.600CH1.62 O0.53 N0.15 +0.400CO2 +0.649H2 O CH2 O+0.090NH3 +0.384O2
EKC271 p. 155/16
Given that the heat of combustion of; 2. anaerobic growth with stoichiometric equation;
0.122CH1.62 O0.53 N0.15 +0.582CH3 O0.5 +0.297CO2 +0.05H2 O CH2 O+0.018NH3
0 Saccharomyces cerevisiae, Hc,cell = 560kJ/mole 0 Glucose, Hc,glucose = 467kJ/mole 0 Ammonia, Hc,ammonia(g) = 383kJ/mole 0 Ethanol, Hc,ethanol = 683kJ/mole
EKC271 p. 156/16
Answer 3: 1. Using equation (27), the heat generated when Saccharomyces cerevisiae is grown aerobically;
Qaerobic =0.600(560)4670.090(383)=165.5kJ/mole Qaerobic =165.5kJ/Cmole glucose
2. Similarly, for the anaerobic growth of yeast;

Qanaerobic =0.122(560)+0.582(683)4670.018(383)=8.1kJ/mole Qanaerobic =8.1kJ/Cmole glucose
EKC271 p. 157/16
Heat Generation by Microbial Growth It is clear from the above results, that the heat generated in the aerobic process is much higher than in the anaerobic process. Large amount of heat is produced when the yeast is grown aerobically is not reected in a correspondingly large biomass yield. This shows that the enthalpy originally present in glucose is wasted in the aerobic process but for the anaerobic process, the enthalpy of glucose is retrieved back in ethanol.
EKC271 p. 158/16
Heat Generation by Microbial Growth It is also apparent that the cooling requirement in aerobic process is much higher compared to anaerobic processes. If bakers yeast is grown aerobically, at a specied growth rate of 0.25hr1 , the specic rate of heat production can be calculated using;
M
rQ =
j=1
Qp,j rp,j
0.25 = 165.5 = 69kJ C mole1 biomass hr1 0.600
EKC271 p. 159/16
Question 3: Heat generation during the batch growth of Saccharomyces cerevisiae: During the batch growth of S. cerevisiae, there is a high glucose concentration at the start of the fermentation, and ethanol is produced. When the glucose is exhausted, the yeast may continue to grow on ethanol, but the specic growth rate is lower and two distinct growth phases are consequently observed: when yeast metabolises glucose it metabolises ethanol
EKC271 p. 160/16
Question 3: Such a growth is known as diauxic growth and can be described by the given stoichiometric equations:
CH1.6 O0.5 N0.15 +2.06CH3 O0.5 +2.20CO2 +1.59H2 O 5.26CH2 O+0.15NH3 +1.13O2
and
CH1.6 O0.5 N0.15 +0.59CO2 +1.81H2 O1.59CH3 O0.5 +0.15NH3 +1.35O2
EKC271 p. 161/16
Question 3: Given that the reaction/growth rate for the rst equation with glucose, glucose = 0.35hr1 and for the second equation with ethanol, ethanol = 0.15hr1 . Calculate the heat of production in each of the two reactions using the heat of combustion given in Example 3.
EKC271 p. 162/16

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BIOTECHNOLOGY FOR ENGINEERS Core Course for B.Eng.

(Chemical Engineering) Semester I (2010/2011)

New Drugs & Clinical Trials

Solid-Liquid Separation Liquid-Liquid Extraction Cell Disruption Solvent Recovery Crystallisation

Gene Modification Gene Expression Recombinant DNA Fermentation Directed Evolution

Thermal heatate of destruction of microbes by r steam/moist heat it is described as;

or upon integration gives,

Higher activation energy, EA

Lower activation energy, EB

A Time (a) High propotion of sensitive culture A

(b) High propotion of sensitive cultur

OR seldom written as;

which later gives;

and upon rearrangement leads to;

if the unsterile culture contains N0 = 1011 viable cells, hence

Design aspects (contd): This can be simplied into;

factor during heating and cooling (contd): Using;

factor during heating and cooling (contd): Therefore,

etc. which gives the summation of individual of;

factor during heating and cooling (contd): Hence,

Holding time (121 C): 1. Holding time can be calculated using

when overall is determined from the given Nt = 103 and N0 = 1011

3. The specic death rate k = 2.54 min1 ,

6. k(T ) is the thermal sterilisation occurred at temp. T in 1 min.

9. If the cooling takes place rapidly in 15 min instead of 21 min, thus,

Continuous plate heat exchange

Continuous steam injection and ash cooling

Batch culture requires enough nutrient to maintain the growth

The rate of cell growth is given by;

Similarly, the rate can be written as;

which upon rearrangement gives;

Taking natural log on both sides of the equation leads to;

A plot of ln x versus time, t gives straight line with a slope .

Taking natural log on both sides;

upon integration leads to

Growth phase Lag

Rate of growth Zero

Comments Inoculum adapting with the changing condition (temperature, pH)

Trivial Population growth changes the environment of the cells

Growth phase Decline

Rate of growth Negative

Cells lyse due to lack of nutrient

Sterile air or oxygen

Sterile air or Nutrient Inlet oxygen

in order to maintain the volume within the vessel;

For a reactor without a recycle system,

let the term leads to,

denote as D, the nal equation

where D is the dilution rate of a CSTR cultivation system.

Material balance: consider the cell concentration in the fermenter;

max [S] , KS +[S]

Material balance: at steady-state,

max [S] =0 KS + [S]

Material balance: consider the substrate/nutrient concentration in the fermenter;

at steady-state condition, [S] = 0 as well.

Material balance: thus;

upon expansion and let =

max [S] KS +[S]

max [S] KS + [S]

Material balance: upon rearranging;

max [S] KS + [S]

for a constant volume in a bioreactor,

Material balance: thus,